Immunoassays, which take advantage of natural immunological reactions, have found widespread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes (called ligands herein) including, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding assays, a labeled ligand analog (sometimes referred to as ligand analog herein) is placed in competition with the unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) ligand analog. The reaction proceeds as follows: ##EQU1##
Useful labels include proteins such as the peroxidases, including horseradish peroxidase (HRP). HRP, for example, has many properties which make it a desirable enzyme for use in enzyme immunoassays including its detectability at low concentrations, a pH optimum compatible with good antigen-antibody binding, long-term stability, low cost and absence of endogenous activity in patient samples.
Many methods for coupling ligands to proteins require the availability of reactive amino groups on the protein. The problem is that proteins such as naturally occurring HRP contain very few reactive amine groups. The number reported varies between 0 and 7 depending on the method of purification used. Thus the methods for coupling enzymes such as HRP to ligands through amine groups results in a significant fraction of unreacted enzyme.